Antibodies play an important role in diagnostic and clinical applications for neutralizing and identifying pathogens. As is known in the art, interaction of the heavy and light chain variable domains (VH and VL) of antibodies, comprising three complementarity-determining regions (CDRs) each, results in the formation of an antigen binding region (Fv). The variable region of an antibody contains the antigen binding determinants of the molecule, and thus determines the specificity of an antibody for its target antigen. This characteristic structure of antibodies provides a stable scaffold upon which substantial antigen-binding diversity can be explored by the immune system to obtain specificity for a broad array of antigens.
The immune repertoire of camelids (camels, dromedaries and llamas) is unique in that it possesses unusual types of antibodies referred to as heavy-chain antibodies (Hamers et al, 1993). These antibodies lack light chains and thus their combining sites consist of one domain, termed VHH. Single domain antibodies (sdAbs) have also been observed in shark and are termed VNARs. Furthermore, recombinant sdAbs may also be prepared using knowledge gleaned from structural studies of naturally-occurring sdAbs.
sdAbs provide several advantages over single-chain Fv (scFv) fragments derived from conventional four-chain antibodies. Single domain antibodies are comparable to their scFv counterparts in terms of affinity, but outperform scFvs in terms of solubility, stability, resistance to aggregation, refoldability, expression yield, and ease of DNA manipulation, library construction and 3-D structural determinations. Many of the aforementioned properties of sdAbs are desired in applications involving antibodies.
Naturally-occurring single domain antibodies can be isolated from libraries (for example, phage display libraries) by panning based solely upon binding property as the selection criterion (Arbabi-Ghahroudi et al., 1997; Lauwereys et al., 1998). However, antibodies/peptides isolated with this method usually have a low to moderate affinity to their antigens/ligands. Since each M13 phage particle presents five copies of the minor coat protein pill, a phage particle displaying an antibody fragment on all copies of pill can be considered a pentavalent antibody. This multivalent display of antibody fragments on phage greatly increases the avidity of the antibody and facilitates both screening and evaluation of phage antibodies. Isolated antibody fragments (scFvs or sdAbs) or peptides bind antigen much less efficiently since they exist primarily in a monovalent form and lack avidity.
An antibody fragment oligomerization strategy that permits pentavalency as in pill phage display is the subject of PCT/CA02/01829 (MacKenzie and Zhang). Fusion of a single domain antibody (sdAb) to the homo-pentamerization domain of the B subunit of verotoxin (VT1B) results in the simultaneous pentamerization of the sdAb. The pentavalent sdAbs, termed pentabodies, bind much more strongly to immobilized antigen than their monomeric counterparts. In the instance of peptide hormone-binding sdAb, pentamerization resulted in 103 to 104-fold improvement in binding to immobilized antigen.
Thus, there is a need in the art to isolate sdAbs that are antigen-specific, soluble and structurally stable for use in clinical and diagnostic applications.